Project Summary Obesity is an extremely costly health problem, largely unresponsive to current therapeutic and prophylactic measures. Heritability estimates indicate that 30-50% of the likelihood of becoming obese is conveyed by genes, and over 100 genes leading to susceptibility to obesity have been identified in rodents and humans. However, the genes so far identified in the aggregate account for no more than ~5% of this risk. Genome wide association studies for obesity are inherently designed to detect common alleles present in 5% or more of the population. We hypothesize that some or most of the ?missing heritability? for obesity is due to rare variants. In Aim 1 we will sequence the exomes (and possibly entire genomes) of individuals and their families in which severe, early-onset obesity is segregating. Multi-tiered bioinformatics filters and pathway analysis will be used to examine the biological functions, molecular networks, and canonical energy homeostasis pathways represented by the prioritized novel variants. In Aim 2?the implicated genes will be characterized in: 1. hypothalamic neurons (and possibly other cell types) created from somatic cells of affected individuals as compared to their isogenic controls (generated using CRISPR); 2. Mice manipulated by direct injection of candidate gene constructs into the brain and mice segregating for knock-in alleles of the candidate gene. These animals will be studied using sophisticated measures of energy homeostasis and food intake, including studies of hedonic aspects of the quantity and quality of good intake. The stem cell-derived neurons will enable study of the developmental, structural, cellular, biochemical/molecular and functional phenotypes of not otherwise accessible for this type of analysis. Success in generating patient-specific hypothalamic neurons from human iPSCs will create a cellular ?reagent? likely to be extremely useful in molecular physiology and drug discovery. The combining of three elements of analysis: exome sequencing/pathway analysis, iPSC- derived hypothalamic neurons/other cells, and creation of animal models subjected to sophisticated metabolic and behavioral phenotyping provides a powerful platform for the identification of novel molecular mechanisms for human obesity. .